Aim 1. Identify critical signaling nodes that integrate RTK and hypoxia signaling pathways in RCC and, where relevant, in other GU malignancies, using HGF/Met as a model system. Progress in the development of prognostic markers of aggressiveness in clear cell renal cell carcinoma (RCC) has accelerated following new insights in molecular pathogenesis afforded by The Cancer Genome Atlas (TCGA) project for this disease . Although pathologic stage and grade are the best available tools at present for stratifying the risk of progression and survival, a significant proportion of cases have a clinical course that does not correlate with predictions. Using information gleaned the TCGA project and database, as well as our own in vitro models of aggressiveness in clear cell RCC, we have performed micro-array mRNA expression profiling analyses designed to identify critical nodes integrating RTK and hypoxia signaling networks. As reported previously, genes regulating cell cycle and division as well as cell-cell interactions were found to be modulated by VHL. However, further analysis using reverse modulation between the three different cell lines according to combinatorial models confirmed that some of these genes may also regulate branching morphogenesis and cell invasion. Surprisingly, the set of modulated genes also comprised multiple candidates derived from the BRCA/Fanconi anemia pathway, which may for the first time establish a link between the latter and the VHL pathway. We have therefore identified a set of genes that may be related to aggressiveness in clear cell RCC and we believe further validation of this set of genes is warranted. These may prove to be promising candidates for outcome and treatment response prognostication, as well as targets for molecular therapeutics strategies. Aim 3: Identify and develop selective natural product inhibitors of HIF2-alpha as therapeutic candidates for the treatment of clear cell RCC. Normoxic stabilization of HIF1-alpha alone, while capable of mimicking some aspects of VHL loss, are not sufficient to reproduce tumorigenesis and in fact, there is evidence that it acts as a tumor suppressor. HIF2-alpha, in contrast, is consistently cast as an oncoprotein. To isolate compounds that selectively modulate HIF2-alpha for use as research tools and drug development leads, a cell-based high throughput screening assay of the NCI Natural Products Repository was developed in collaboration with Drs. Tawnya McKee and James McMahon of CCR's Molecular Targets Laboratory (MTL). A series of plasmids were engineered containing tandem copies of the minimal hypoxia response element of the VEGFA gene promoter upstream of a Luc reporter. These plasmids were transfected into the VHL- and HIF1-alpha-negative ccRCC cell line 786-0 and derived cell lines were optimized for use in high-throughput detection systems in the MTL. Secondary counter screens were included for global transcriptional repression and cell toxicity. Leads from Natural Products Repository screening were chromatographically separated into component structures yielding 40 pure compounds with micromolar or submicromolar IC50 values, 80% inhibition and 10% cell toxicity. Lead compounds have been further characterized for HIF1-alpha and HIF2-alpha selectivity using clear cell RCC-derived cell lines engineered to express either factor alone or both. Cells were exposed to lead compounds and modulation of a panel of HIF gene targets with known resposiveness to either factor or both was measured by qRT-PCR. In addition, docking studies using the compounds and the HIF1-alpha and HIF2-alpha crystal structures are in progress to characterize target engagement and support future structure-activity relationship experiments.